Aflatoxin B1, produced by certain strains of Aspergillus flavus, is a potent liver carcinogen for many animal species including, possibly man. Recent studies from this laboratory have demonstrated this carcinogen, when injected into rats, strongly inhibits rat liver nucleolar RNA synthesis (J. Biol. Chem. 252, 3245, 1977). Furthermore, it was found that the enzyme, RNA polymerase I, which transcribes the nucleolar chromatin was not affected by this carcinogen. In order to understand the mechanism of this inhibition, other alternatives should be considered: a) aflatoxin B1 may interact with nucleolar DNA thus inpairing its template function, or b) aflatoxin B1 may react with one of the key regulatory proteins on the nucleolar chromatin and, as a result, the expression of the nucleolar gene is greatly inhibited. It is the aim of this proposal, therefore to determine which one of the above mentioned alternatives is the true mechanism of action of aflatoxin B1 in the inhibition of nucleolar RNA synthesis. In order to determine whether the nucleolar DNA template is the source of inhibition, experiments will be carried out using RNA polymerase I from control group to transcribe a) isolated total nucleolar DNA, b) enriched rDNA gene, c) purified rDNA gene from both control and aflatoxin B1 treated groups, to see if the DNA template function from aflatoxin B1 treated group is indeed impaired. In order to determine whether nucleolar chromosomal proteins are directly involved in the aflatoxin B1 inhibition, nucleolar histones, nucleolar High Mobility Group proteins, and other non-histone proteins are selectively extracted from control and aflatoxin B1 treated nucleolar chromatin. These proteins from each group are then used in step-wise fashion to reconstitute nucleolar chromatin with identical nucleolar DNA from the control source. RNA polymerase I isolated from control group will be used to transcribe the chromatin template at each stage of the reconstruction, thus to ascertain if, indeed, certain groups of proteins from the aflatoxin B1 treated group produces clear differential inhibitory effect on the otherwise normal nucleolar DNA template. The protein involved can be identified by further purification.